-Previously, we developed a highly sensitive assay for viremia, capable of detecting a single copy of HIV-1 RNA in plasma. Studying individuals with HIV suppressed by antiretroviral therapy, we found that viremia in patients is independent of regimen but strongly associated with pretherapy virus levels, implying that, even after 7 years of treatment cells infected prior to therapy survive to make more virus. Along with R. Siliciano we completed a clinical trial demonstrating the lack of additional suppression of HIV-1 viremia on intensification of standard antiretroviral therapy with either protease inhibitor or NNRTI intensification. During the past year we extended these studies in an additional intensification trial with the new integrase inhibitor, raltegravir. Along with D. McMahon we demonstrated no evidence of additional suppression by raltegravir intensification. Along with ACTG investigators, R. Gandhi, PI, we participated in a large multisite, randomized study of raltegravir intensification using a longer duration of intensification. This study confirmed our pilot study and demonstrated that prolonged intensification didn't result in additional decline in viremia. Taken together these experiments have provided strong evidence that the source of persistent viremia during antiretroviral suppression is not active cycles of HIV replication in short lived CD4 T cells. -These natural history and interventional studies indicate that new approaches are needed to eradicate HIV-1 infection. Several new projects have been initiated: 1)We have started a new protocol (11-I-0057) to investigate the effect of interferon alpha in suppressing persistent viremia in patients suppressed on standard antiretroviral therapy. 2) To investigate the potential for cytotoxic chemotherapy to reduce the levels of HIV production from cells capable of cellular expansion, we are collaborating with R. Yarchoan (NCI HIV and AIDS Malignancy Branch) in a study of administration of cytotoxic chemotherapy and local radiation therapy for HIV associated anal neoplasms (11-C-0129). Adapting the intensification model, patients with suppressed viremia are undergoing additional phlebotomy to obtain plasma and PBMC prior to, during and following cycles of chemotherapy with the alkylating agent 5-fluorocuracil and DNA crosslinking agent mitomycin C (N=15). Additionally patients will be sampled during and following subsequent periods of radiation. Our hypothesis is that by suppressing cell division, alkylating and crosslinking agents will result in decreases in HIV viremia, especially in patients with PPC but local radiation will not result in changes in HIV RNA. We'll analyze HIV population genetics from plasma, PBMC, and from GALT obtained from regions proximal and distant to the tumor tissue to determine whether changes in viremia can be mapped to specific reservoirs. 11-C-0129 is open at NIH and is currently accruing patients. 3) We are working with E. Reid to measure effects of the proteasome inhibitor, bortezomib, on persistent HIV-1 viremia in infected individuals with suppressed viemia who are receiving bortezomib as therapy for co-morbid lymphoma (Bench to Bedside 2008). These studies will shed light on potential new directions designed to eradicate HIV-1 infection in individuals suppressed by antiretroviral therapy. -We have initiated new studies to investigate the characteristics of low level viremia, as well as new analyses to investigate the establishment of low level viremia. With M. DiMascio (NIAID) we are investigating immune correlates of 1st and 2nd phase decay kinetics in a cohort of well characterized patients followed at NIH for prolonged periods (&gt;8 y, NIH protocol 97-I-0082, now 08-I-0221). In initial analyses we identified significant variability in phase II decay kinetics and are investigating immune correlates (CD4, CD8, CD45RO/RA cell numbers) responsible for variability. Also with M. King we are investigating immune correlates of persistent low level viremia in individuals suppressed for prolonged periods. In initial studies we found that patients in 08-I-0221 who have been suppressed for &gt;8 years still have small but statistically significant increase in immune activation when compared to uninfected individuals. We also detected a correlation between the level of persistent viremia and cellular immune activation. To better understand mechanisms of persistence and the role of immune responses in viral suppression, we are performing an integrated analysis quantifying cellular and soluble immune activation markers and HIV viremia in a well-characterized cohort of 44 suppressed patients at NIH (Protocol 97-I-0082/08-I-0221). Patients enrolled in this study have had extensive immunophenotyping and have stored plasma and lymphocytes from before ART and through 8-10 years of combination ART. -During the past several years, a number of independent lines of evidence have suggested a strong contribution of gut associated lymphoid tissue (GALT) to HIV replication. Defects in gut function as a result of HIV infection result in increased translocation of bacterial components into blood, a process which has been proposed to drive a chronic inflammatory state, increase T cell activation, and virus production from chronically infected cells. To investigate the role of bacterial translocation in persistent viremia we are investigating the effects of reductions in bowel flora in HIV persistent viremia. Specifically we are investigating the effects of the non-absorbable antibiotic, rifaxamin, on bacterial translocation, immune activation, and persistent viremia. We received a Bench to Bedside Award (2009) and will be collaborating with D. McMahon and A. Ganesan. -The single copy analytic technique is being utilized as a benchmark for the development of new approaches to measure HIV-1. With I. Hewlett and L. Demeter we are participating in development of europium based p24 detection assays (Bench to Bedside 2008). -The same single copy assay is being used (with B. Walker) to probe the levels of viremia in the rare HIV-1-infected patients who are able to control their viremia at very low levels in the absence of therapy, as well as in a well-described NIH cohort of patients with HLA-restricted HIV-1 replication (with S. Migueles, M. Conners, and H.C. Lane) and also (with D. Margolis) to see whether a drug that activates HIV expression (i.e., Suberoylanilide hydroxamic acid, SAHA) can affect the level of persistent virus. -We are expanding the utility of the single copy assay to to quantitate cell associated HIV nucleic acid. With J. Mellors, we are developing techniques to measure both HIV RNA and DNA, including new assays to measure circular forms of HIV DNA. These assays will be useful in investigating the course of HIV replication both in peripheral blood derived cells as well as tissue derived material. -We are investigating the nature of HIV-1 replication during suppressive therapy in specific patient populations. We have initiated new collaborations with J. Ananworanich and I. Sereti to investigate HIV-1 subtype A/E viremia in patients suppressed on antiretroviral therapy who initiated therapy during acute seroconversion. [Corresponds to Project 1 in the April 2007 site visit report of the Host-Virus Interaction Branch, HIV Drug Resistance Program]